H. M. Pinsker

Bag Cell Control of Egg Laying in Freely Behaving Aplysia

Neuroendocrine (bag cell) control of egg laying was studied in freely behaving Aplysia. Surgical lesions showed that bag cells are not necessary for egg laying, although they play a crucial role in its control, and that the pleurovisceral connectives are the afferent pathway to the bag cells. Recording in vivo showed that synchronous bag cell spikes progressively invade the network, leading to prolonged repetitive firing that initiates natural egg laying.

Computer separation of unitary spikes from whole-nerve recordings.

A practical and efficient off-line computer technique is described for automatically separating unitary waveforms from multiunit whole-nerve spike train data with no a priori knowledge about the number of units or their waveforms. The procedure requires two recording eletrodes which provide 3 measures on each putative unit: (1) peak-to-peak amplitude on the proximal channel, (2) amplitude on the distal channel, and (3) temporal offset (depends on conduction velocity) between proximal and distal spikes. On the basis of these 3 measurements, individual unitary spikes are automatically separated into clusters according to empirically-determined limits of variability. The results of the program are displayed in 3-D plots of the 3 measures on each unitary spike and in plots of superimposed waveforms from each cluster. These plots can be used to interactively correct clustering errors. The procedure is illustrated with a 1-min segment of spike train data recorded in vivo from the siphon nerve of a freely-behaving Aplysia. We routinely obtain about 10 relatively well-isolated units in such segments. By utilizing the average waveforms and conduction velocities for individual clusters, it may eventually be possible to separate unitary spikes from compound waveforms resulting from simultaneous of two or more units.

Phase plane description of endogenous neuronal oscillators in Aplysia

Phase plane techniques are used to describe graphically the limit cycle behavior of identified endogenous neuronal oscillators in the isolated abdominal ganglion of Aplysia. Intracellularly recorded membrane potential from a bursting neuron and its first derivative with respect to time are used as coordinates (state variables) in phase space. The derivative is either measured electronically or calculated digitally. Each trajectory in phase space represents the entire output of the bursting neuron, i.e., both the rapid action potentials and slow pacemaker potentials. Phase plane portraits are presented for the free run limit cycle before and after a change in a system parameter (applied transmembrane current) and also for phase resetting produced by direct synaptic inhibition from an identified interneuron. The complex topology of the trajectory suggests that the bursting oscillator is a higher order system. Therefore, the second time derivative is used as another state variable. This type of phase plot can help to relate biophysical and mathematical analyses.

Temperature dependence of egg laying inAplysia brasiliana andA. californica

SummaryThe temperature dependence of egg laying was examined in winter-caughtAplysia. Cold-waterAplysia californica and warm-waterA. brasiliana were individually housed in the same large aquarium for 16 days at 15°C, and then for 16 days at 20°C. Initially, the majority of theA. californica were not reproductively mature (as determined by injections of atrial gland extracts) whereas all of theA. brasiliana were reproductively mature. When the temperature was increased from 15 to 20°C, both species showed a marked increase in the frequency of egg laying. At both temperatures,A. brasiliana laid eggs more frequently but produced smaller egg masses thanA. californica. We conclude that increased egg laying inA. californica was attibutable both to facilitation of oogenesis in previously reproductively immature animals and to increased activity of the bag cells which release an egg-laying hormone. Increased egg laying inA. brasiliana was attributable primarily to increased bag cell activity.

Role of bag cells in egg deposition ofAplysia brasiliana

SummaryThe spontaneous discharge of the neurosecretory bag cells causes ovulation in the marine gastropod,Aplysia brasiliana, but the overt head movements that subsequently deposit the eggs on the substrate are initiated before the bag cells fire (see accompanying paper). Injection of homogenized abdominal ganglia (HAG) containing bag cells into intact animals triggers (1) release of eggs from the ovotestis, (2) similar head movements associated with egg deposition and (3) a sustained period of inhibited feeding. We now report that the full expression of the overt behavioral effects of HAG injections are influenced by egg movement through the reproductive tract. When egg movement is blocked at the small hermaphroditic duct (SHD) near the ovotestis, HAG injected animals show significantly decreased head oscillations compared to mock operated controls. Furthermore, SDH ligated animals do not stop responding to food after HAG injections, unlike mock operated animals that show complete inhibition of feeding. Thus movement of eggs through the reproductive tract beyond the SHD is required for normal head movements and feeding inhibition. Artificial “egg” movement through a cannula in the reproductive tract indicates that neither HAG injection nor movement of artificial “eggs” alone triggers head movements effectively. However, the combination of HAG and artificial “eggs” triggers behaviors quantitatively more similar to those with normal egg movement. Finally, denervation of the reproductive tract does not prevent the normal occurrence of head movements following HAG injection when eggs are allowed to move normally. These results suggest the hypothesis that the release of bag cell hormones and the subsequent movement of eggs normally maintain the previously initiated head movements so that egg laying behaviors stop when all of the eggs are deposited.

Egg laying inAplysia

SummaryAplysia egg laying is a complex sequence of head and neck movements initiated by the release of ovulatory and neuroactive hormones from the neurosecretory bag cells. This behavioral pattern is difficult to study in reduced preparations, because they do not show ovulation or egg laying behaviors. This paper describes the use of chronically implanted electrodes to elicit normal neurosecretory activity and provides an analysis of egg laying behaviors and the underlying muscle activity in intact, freely behavingA. californica andA. brasiliana.1.Bag cell discharges elicited with a fine wire electrode implanted in the connective tissue sheath above the cell bodies were typically without noxious behavioral side effects.2.Following selectively elicited bag cell discharges, egg laying consisted of four rhythmic head and neck movements that were separated functionally into appetitive behaviors (‘waves’ and ‘undulations’) used to explore and prepare the substrate and consummatory behaviors (‘weaves’ and ‘tamps’) used to distribute and attach the egg string. The amount of time an animal performed consummatory behaviors was positively related to the amount of eggs deposited. By contrast, the appetitive phase of egg laying was independent of the size of the egg mass.3.The individual behaviors and their temporal sequence were similar following selectively elicited bag cell discharges, spontaneous discharges of animals with implanted electrodes and during normal egg laying of unoperated animals.4.Three longitudinal muscle systems occurred within the head and neck. Following a selectively elicited bag cell discharge, spatially and temporally coordinated patterns of EJP bursts of different durations were recorded chronically from each muscle group. These EJP patterns were characteristic for specific head and neck movements used in appetitive and consummatory egg laying behaviors.

Bradycardial response inAplysia exposed to air

SummaryHeart rate was chronically monitored (Figs. 1, 3) in two species of the marine gastropodAplysia. The warm waterA. brasiliana have an average basal heart rate in water of 33 min−1, whereas the cold waterA. californicas heart rate is 20.6 min−1. The heart rate in both species shows a strong temperature dependence and the difference in basal heart rate is negligible when measured at the same temperature (Fig. 2). Both species show a consistent bradycardia when exposed to air (Fig. 4):A. brasiliana showed a 43% average decrease in air, whereasA. californica showed only a 16.5% decrease. Removal of the abdominal ganglion produced no significant decrease in heart rate in either species, nor did it reduce the bradycardial response to air exposure inA. californica (Fig. 8). However, it significantly reduced, but did not abolish, the bradycardia inA. brasiliana (Figs. 5, 6, 7). We conclude that the bradycardia has a significant central component inA. brasiliana, but is peripherally mediated inA. californica. The bradycardial response to air exposure may be analogous to the diving response in air breathing vertebrates.

Environmental modulation of a central pattern generator in freely behaving Aplysia..

A stereotyped contraction of the gill, siphon, mantle shelf, and parapodia of Aplysia californica is mediated by a central pattern generator, the Interneuron II network. Activity of the central pattern generator was monitored with chronically implanted electrodes on the siphon nerve of intact animals. A characteristic patterned burst of activity in siphon motoneurons is associated with spontaneous Interneuron II contractions and is clearly different from heightened motor activity associated with other siphon movements. The Interneuron II contraction can also be modulated by tactile stimulation of the siphon and other environmental stimuli. We examine the time course of effects of siphon stimulation, and show that changes in illumination and feeding cause longer lasting modulation of burst frequency.

Non‐associative learning in the squid lolliguncula brevis (Mollusca, Cephalopoda)

Habituation and dishabituation are demonstrated for the first time in a squid (Cephalopoda: Teuthoidea). Each squid (n = 29) was exposed briefly to a plastic model of a predator to determine how the behavioural responses changed with repeated trials at 1 min intervals. Behavioural responses were video‐monitored for subsequent measurements of the number of escape jets and the duration of neurally controlled body pattern rings on each trial. Squids habituated readily to a teleost fish model and could differentiate clearly between a teleost fish and a shark model of the same size. Following a single series of 15 trials, habituated responses recovered after a 1 hr rest. Habituated responses also recovered rapidly (dishabituation) when a noxious stimulus was presented. Spaced Training was more effective than Massed Training in producing longer‐term habituation.